U.S. patent application number 12/994936 was filed with the patent office on 2011-05-26 for compositions and methods for spatial separation and screening of cells.
This patent application is currently assigned to Massachuesetts Institute of Technology. Invention is credited to J. Christopher Love, Kerry Love.
Application Number | 20110124520 12/994936 |
Document ID | / |
Family ID | 41377450 |
Filed Date | 2011-05-26 |
United States Patent
Application |
20110124520 |
Kind Code |
A1 |
Love; J. Christopher ; et
al. |
May 26, 2011 |
Compositions and Methods for Spatial Separation and Screening of
Cells
Abstract
The invention provides a method for isolating particular members
from a library of variant cells in individual microreactors,
wherein the phenotype of the biomolecule secreted by the cell is
evaluated on the basis of multiple parameters, including substrate
specificity and kinetic efficiency.
Inventors: |
Love; J. Christopher;
(Somerville, MA) ; Love; Kerry; (Somerville,
MA) |
Assignee: |
Massachuesetts Institute of
Technology
Cambridge
MA
Whitehead Institute for Biomedical Research
Cambridge
MA
|
Family ID: |
41377450 |
Appl. No.: |
12/994936 |
Filed: |
June 1, 2009 |
PCT Filed: |
June 1, 2009 |
PCT NO: |
PCT/US2009/003354 |
371 Date: |
January 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61057371 |
May 30, 2008 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/11;
506/7 |
Current CPC
Class: |
C12Q 1/34 20130101; C12Q
1/533 20130101; C12Q 1/26 20130101; G01N 2333/91091 20130101; C12Q
1/02 20130101; C12Q 1/37 20130101; C12Q 1/25 20130101; C12Q 1/527
20130101; G01N 33/5005 20130101 |
Class at
Publication: |
506/9 ; 506/7;
506/11 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 30/00 20060101 C40B030/00; C40B 30/08 20060101
C40B030/08 |
Claims
1. A method of performing solution-phase biomolecule screening,
comprising: depositing a library of cells onto a microdevice,
wherein said microdevice contains wells that spatially separate
said cells in solution, wherein said cells are distributed about
one cell per well, wherein a plurality of cells secrete variants of
at least one biomolecule in said solution; contacting said secreted
biomolecule variants with at least one optical signal substrate,
each indicative of a desired biomolecule phenotype or activity;
evaluating the phenotype of the biomolecule encoded by the cell on
the basis of multiple parameters, and isolating said cells that
secrete a desired biomolecule variant from said microdevice.
2. The method of claim 1, wherein said phenotype is evaluated by
detecting changes over time in one or more optical signals
generated by one or more optical signal substrates in the library
of cells, wherein such changes indicate a desired biomolecule
phenotype or activity of the variants of the biomolecule.
3. The method of claim 2, wherein said optical signal is a
fluorescence signal.
4. The method of claim 3, wherein said biomolecule phenotype or
activity is monitored in real-time or near-real-time in said
microdevice on the basis of changes in the intensities of said
fluorescent signal.
5. The method of claim 1, wherein said biomolecule is selected from
the group consisting of a peptide, a polypeptide, a protease, an
oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase,
a ligase, an enzyme, an antibody, a cytokine, a chemokine, a
nucleic acid, a metabolite, a small molecule (<1 kDa) and a
synthetic molecule.
6. The method of claim 5, wherein the molecular weight of said
biomolecule is greater than about 600 Da and less than about
100,000 Da.
7. The method of claim 1, wherein said parameters are selected from
the group consisting of catalytic rate, specificity of reaction,
kinetic efficiency, and substrate binding affinity.
8. The method of claim 7, wherein said parameters are evaluated in
parallel.
9. The method of claim 1, wherein said cells are eukaryotic
cells.
10. The method of claim 9, wherein said eukaryotic cells are yeast
cells.
11. The method of claim 1, wherein said wells are between about 10
and 100 .mu.m in diameter.
12. The method of claim 1, wherein said cells are isolated by
micromanipulation with a glass capillary.
13. The method of claim 12, further comprising randomly
mutagenizing said biomolecule.
14. The method of claim 12, further comprising sequencing said
biomolecule.
15. The method of claim 1, wherein said biomolecule is a mutant
glycosyltransferase (GTase) or a glycosidase.
16. The method of claim 15, wherein said GTase is capable of
competing with chemical synthesis for the rapid and large scale
production of complex carbohydrates.
Description
RELATED APPLICATIONS
[0001] This application is a national stage application, filed
under 35 U.S.C. 371, of International Application No.
PCT/US2009/003354, filed Jun. 1, 2009, which claims the benefit of
provisional application U.S. Ser. No. 61/057,371, filed May 30,
2008, the contents of which are incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The invention provides a method for isolating particular
members from a library of variant cells in individual
microreactors, wherein the phenotype of the biomolecule encoded by
the cell is evaluated on the basis of multiple parameters,
including substrate specificity and kinetic efficiency.
BACKGROUND OF THE INVENTION
[0003] Enzymes are increasingly being used as catalysts in
industry, agriculture, medicine and scientific research. Due to
their substrate specificity, chemical selectivity and environmental
compatibility, enzymes offer advantages for such applications as
the synthesis of chirally pure pharmaceuticals, textile processing,
food processing, medical diagnostics and therapy, biotransformation
and bioremediation. Enzymes are proving to be superior to
traditional chemical processes for modifying high molecular weight
polymers.
[0004] Evaluation of libraries of genetic variants of biomolecules,
such as enzymes, to identify specific members in the library with
desired properties requires both characterizing the phenotype of
the biomolecule produced and correlating the biomolecule to the
genotype of the member of the library encoding it. In this way,
desired variants are selected and further evaluated. Directed
evolution has proven particularly successful in cases where enzyme
function is directly linked to cell survival, i.e., restoration of
an essential activity that has been deleted from an otherwise
wild-type cell. However, evolution of enzymes that do not
themselves provide a selectable phenotype, as in the case of
glycosyltransferases (GTases) and other transferases, is much more
difficult. While selection strategies do exist to evolve enzymes of
this sort, including chemical complementation, phage display and
bacterial cell surface display, current methods do not provide a
facile or generalized strategy for engineering diverse enzymes. As
the demand for new biomolecules grows, there is a pressing need for
new strategies for engineering enzymes with improved activity and
novel catalytic function.
SUMMARY OF THE INVENTION
[0005] The invention provides methods for isolating particular
members from a library of variant cells in individual
microreactors, wherein the phenotype or activity of the biomolecule
encoded by the cell is evaluated on the basis of multiple
parameters, including substrate specificity and kinetic
efficiency.
[0006] In one aspect, the invention relates to compositions and
methods for screening libraries of secreted products for novel
phenotypes, including enzymes with improved catalytic properties or
altered substrate specificity using microwells for the special
separation of cells producing the enzymes.
[0007] In another aspect, the invention provides for methods of
performing biomolecule screening in solution phase, e.g., directed
evolution biomolecule screening, comprising depositing a library of
cells onto a microdevice, wherein the microdevice contains a
plurality of wells that spatially separate the cells in solution.
The cells are distributed at about one cell per well, and a
plurality of cells secrete variants of at least one biomolecule in
the solution. The secreted biomolecule variants are contacted with
at least one optical signal substrate, each indicative of a desired
biomolecule phenotype or activity; and the phenotype of the
biomolecule encoded by the cell is evaluated on the basis of
multiple parameters. In some cases, the "optical signal substrate"
is a composite of one or more units, e.g., an antibody or other
specific ligand or small molecule tag that is directly conjugated
to a detectable marker. For example, in a two element reaction
(e.g., X+Y catalyzed by a transferase enzyme), a first element,
"Y", is captured by an antibody or other ligand that is immobilized
on a surface such as a culture plate and the second element, "X",
is detected with an optical substrate such as a
fluorescently-tagged antibody. The cells that secrete a desired
biomolecule variant from the microdevice are then isolated.
[0008] Optionally, the phenotype is evaluated by detecting changes
over time in one or more optical signals generated by one or more
optical signal substrates in the library of cells, wherein such
changes indicate desired biomolecule phenotype or activity of the
variants of the biomolecule. The invention utilizes various
chromogenic, fluorogenic, lumigenic and fluorescence resonance
energy transfer (FRET) substrates to measure biological activity.
Many donor/acceptor FRET pairs are commercially available. These
include, but are not limited to: 5-carboxytetramethylrhodamine
(TAMRA)/QSY-7 (diarylrhodamine derivative); Dansyl/Eosin;
Tryptophan/Dansyl; Fluorescein/Texas Red (rhodamine);
Naphthalene/Dansyl; Dansyl/octadecylrhodamine (ODR);
boron-dipyrromethene (BODIPY)/BODIPY; Terbium/Thodamine;
Dansyl/fluorescein isothiocyanate (FITC); Pyrere/Coumarin;
5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid
(IAEDANS)/IAFBPE/Cy5; and Europium/Cy5. Preferably, the optical
signal is a fluorescence signal. In one aspect, the biomolecule
phenotype or activity is monitored in real-time or near-real-time
in the microdevice on the basis of changes in the intensities of
the fluorescent signal.
[0009] The invention provides that the biomolecule is selected from
the group consisting of a secreted molecule, a peptide, a
polypeptide, an enzyme such as a protease, an oxidoreductase, a
transferase, a hydrolase, a hydrogenase, a lyase, an isomerase, a
ligase, a polymerase, as well as an antibody, a cytokine, a
chemokine, a nucleic acid, a metabolite, a small molecule (<1
kDa) and a synthetic molecule. For example, the molecular weight of
the biomolecule is greater than about 100 Da and less than about
100,000 Da. Alternatively, the molecular weight of the biomolecule
is greater than about 600 Da and less than about 30,000 Da; greater
than about 800 Da and less than about 10,000 Da; or greater than
about 900 Da and less than about 1,000 Da.
[0010] In one approach, activity of the enzyme biomolecule is
evaluated by detecting the proximity of two or more elements upon
which the enzyme or other biomolecule acts. For example, the enzyme
brings together the elements (e.g., ligase) or separates the
elements (e.g., lyase). As described above, detection is
accomplished using FRET pairs or a capture based assay in which a
first element is biotinylated (and captured with an avidin-based
reagent) and a second element is labeled with a fluorescent tag. An
increase or decrease in the association of the elements
(substrates) reflects altered binding specificity/activity of the
enzyme.
[0011] The invention provides for evaluating the phenotype of the
biomolecule encoded by the cell on the basis of multiple
parameters, wherein the parameters are selected from the group
consisting of catalytic rate, specificity of reaction, kinetic
efficiency, and substrate binding affinity. In another aspect, rate
or substrate tolerance, and pH or temperature tolerance are
evaluated. Preferably, the parameters are evaluated in
parallel.
[0012] The invention provides for screening biomolecules secreted
by cells. In one aspect, the cells are eukaryotic cells.
Preferably, the eukaryotic cells are yeast cells. Alternatively,
the cells are prokaryotic cells.
[0013] The invention also provides for a microdevice that contains
wells that spatially separate the cells in solution, e.g., each
well contains solely a single cell. Preferably, the wells are
between about 10 and about 100 .mu.m in diameter, e.g., 10 .mu.m,
20 .mu.m, 30 .mu.m, 50 .mu.m, or 75 .mu.m in diameter.
[0014] In one aspect, the invention provides for isolating the
cells that secrete a desired biomolecule variant from the
microdevice. Preferably, the cells are isolated by
micromanipulation with a glass capillary. Optionally, the invention
provides for randomly mutagenizing the desired biomolecule for
further selection. Suitable techniques for random mutagenesis
include error-prone polymerase chain reaction (PCR), codon cassette
mutagenesis, deoxyribonucleic acid (DNA) shuffling, staggered
extension process (StEP), chemical mutagenesis and the use of
mutator strains. Alternatively, the biomolecule is sequenced to
identify the biomolecule.
[0015] Biomolecules to be interrogated include enzymes. For
example, the biomolecule is a mutant glycosyltransferase (GTase), a
carbohydrate processing enzyme, a carbohydrate binding protein, a
glycosidase, or a lectin affinity protein that binds carbohydrates.
Preferably, the GTase is capable of competing with chemical
synthesis for the rapid and large scale production of complex
carbohydrates. Alternatively, the biomolecules are cytokines,
chemokines, antibodies, or other secreted cell metabolites.
[0016] In yet another aspect, the invention provides for directed
evolution of existing GTases to identify more potent catalysts with
altered substrate selectivity. More specifically, the invention
provides for the identification of mutant GTases capable of
competing with chemical synthesis for the rapid and large scale
production of glycoconjugates for therapeutic purposes, including
carbohydrate-based cancer vaccines and carbohydrate-containing
antibiotics.
[0017] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims. All references cited
herein are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic illustration of a method for
identifying enzymes with new or improved function. Yeast cells
secrete proteins of interest within the microreactors. As every
cell is contained within its own well, each well corresponds to a
single library member. Following the screening of the invention,
the cells are retrieved and used either in further rounds of
screening or for identification of the encoded protein.
[0019] FIG. 2 is a schematic illustrating mucin-type O-linked
glycans.
[0020] FIG. 3 is a schematic illustrating substrates for the
detection of tobacco etch virus (TEV) protease catalytic activity
containing a dipyrromethene boron difluoride (BODIPY) fluorophore
(F) and a tetramethylrhodamine (TAMRA) quencher (Q).
[0021] FIG. 4 is a schematic showing substrates for the detection
of ppGalNAcTase-T1 catalytic activity.
[0022] FIG. 5 is a series of diagrams; (A) is a schematic
illustrating an exemplary antitumor vaccine; (B) is a schematic
showing an exemplary antiparasitic vaccine; (C) is a schematic
illustrating an exemplary antimicrobial vaccine; and (D) is a
schematic illustrating an exemplary antimicrobial agent.
[0023] FIG. 6 is a diagram that demonstrates the structural
comparison of the following glycosyltransferases: BTG, MurG, and
GtfB.
[0024] FIG. 7 is a schematic illustration of directed evolution for
enzyme engineering and catalyst development.
[0025] FIG. 8A is a schematic illustration of a method for
correlating proteins with the cells that secrete them, in which
substrates/products are captured on the contacted glass surface;
(B) is a photograph of a device containing wells between 50 and 100
.mu.m in diameter; (C) is a photomicrograph of a protein microarray
of secreted products from Pichia pastoris cells; and D) is a
photomicrograph of Pichia pastoris cells in microwells.
[0026] FIG. 9 is a photomicrograph of a standard curve for the
comparison of protein secretion levels between different cell
typres, such as hybridomas, Pichia pastoris, and cytokine-secreting
peripheral blood mononuclear cells (PBMC).
[0027] FIG. 10 is a series of photomicrographs demonstrating cell
retrieval using a micromanipulator.
[0028] FIG. 11 is a diagram showing a method for detecting enzyme
turnover in microwells via a trypsin cleavage assay.
[0029] FIG. 12 is a series of photomicrographs demonstrating
fluorescent signal intensity after increasing concentrations (0.05
.mu.g/ml, 0.5 .mu.g/ml, and 5 .mu.g/ml) of trypsin were incubated
with 10 .mu.g/ml FTC-casein for 1 hour in microwells.
[0030] FIG. 13 is a series of photomicrographs depicting
fluorescent signal intensity after 0.5 .mu.g/ml of trypsin was
incubated with 10 .mu.g/ml FTC-casein for 1 and 18 hours in
microwells.
[0031] FIG. 14 is a diagram showing a method for detecting enzyme
turnover in microwells via an HRV-3C protease assay.
[0032] FIG. 15 is a series of photomicrographs showing the results
of the HRV-3C protease assay after incubation in 100 .mu.g/ml FRET
peptide in 1.times. reaction buffer containing media (YPD media)
for 18 hours at room temperature (RT).
DETAILED DESCRIPTION OF THE INVENTION
[0033] Due to their substrate specificity, chemical selectivity and
environmental compatibility, enzymes offer advantages for such
applications as the synthesis of chirally pure pharmaceuticals
useful in medical diagnostics and therapy. Indeed, such enzymes are
utilized in the synthesis of oligosaccharides and glycoconjugates,
which have diverse medical applications, including antitumor
vaccines (targeting, e.g., GM3, a melanoma-related
glycosphingolipid), antiparasitic vaccines (targeting, e.g.,
malarial glycosylphosphatidylinositol (GPI anchor), antimicrobial
vaccines (targeting, e.g., capsular polysaccharide antigen
Haemophilus influenzae serotype b (HIB)), and other antimicrobial
agents. Exemplary antitumor vaccines, antiparasitic vaccines,
antimicrobial vaccines, and antimicrobial agents are shown in FIGS.
5A-5D, respectively. Use of glycosylated biomolecules requires not
only intimate knowledge of structural and functional relationships,
but also access to defined structures for large scale clinical
use.
[0034] Although many wild-type enzymes (i.e., those whose amino
acid sequences are the same as those found in naturally occurring
organisms) can be used without any modification, there are many
instances wherein the physical properties of an enzyme or its
chemical activity are not compatible with a desired application.
Novel physical properties which might be desirable could include,
for example, thermal stability, resistance to non-aqueous solvents,
salt, metals, inhibitors, proteases, extremes of pH and the like.
Reducing the size of the enzyme, abolishing its dependence on
cofactors or other proteins, improving its expression in the host
strain and other similar changes might also be desirable for a
particular application. Improved chemical activities might include,
for example, enhanced catalytic rate, substrate affinity and
specificity, regioselectivity, enantioselectivity, reduced product
inhibition, or an altered pH-activity profile. In addition, it may
be desirable to alter the properties of one or more enzymes that
function together as part of a metabolic pathway.
[0035] As the demand for enzymes with improved activity and novel
catalytic function grows, new methods have been developed for
isolation of a desired catalyst from a pool of protein variants.
Directed evolution has proven particularly successful in cases
where enzyme function is directly linked to cell survival, i.e.,
restoration of an essential activity that has been deleted from an
otherwise wild-type cell. Evolution of enzymes that do not
themselves provide a selectable phenotype, as in the case of
glycosyltransferases (GTases) and other transferases, is much more
difficult. Prior to the invention described herein, no method
provided a facile or generalized strategy for engineering diverse
enzymes.
[0036] The isolated biomolecules are purified naturally-occurring,
synthetically produced, or recombinant compounds, e.g.,
polypeptides, nucleic acids, small molecules, or other agents.
Purified compounds are at least 60% by weight (dry weight) the
compound of interest. Preferably, the preparation is at least 75%,
more preferably at least 90%, and most preferably at least 99%, by
weight the compound of interest. Purity is measured by any
appropriate standard method, for example, by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. By "purified"
or "substantially purified" is meant a biomolecule or biologically
active portion thereof that is substantially free of cellular
material or other contaminating macromolecules, e.g.,
polysaccharides, nucleic acids, or proteins, from the cell or
tissue source from which the biomolecule is derived. The phrase
"substantially purified" also includes a biomolecule that is
substantially free from chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of biomolecules that are
separated from cellular components of the cells from which it is
isolated.
Directed Evolution for Enzyme Engineering and Catalyst
Development
[0037] A schematic illustration of the directed evolution for
enzyme engineering and catalyst development invention is shown in
FIG. 7. The invention provides for the ability to coax/generate
novel activity from an existing enzyme scaffold by iterative rounds
of mutagenesis and selection. As described in detail below, there
are many techniques for randomly mutagenizing the desired
biomolecule for further selection or screening. There are also many
suitable methods for selection and screening. Those skilled in the
art will understand that a specific technique can be chosen based
on the amount of structural information available for the
biomolecule, e.g., protein, of interest. When selecting an
individual technique, it is crucial to maintain a link between
genotype and phenotype, while maintaining high-throughput.
Screening Strategy
[0038] The invention described here provides an automatable,
high-throughput method of evaluating the phenotype of a biomolecule
encoded by a cell on the basis of multiple parameters, including
substrate specificity and kinetic efficiency. This general strategy
allows for the ex vivo screening of diverse enzymes using native or
minimally perturbed substrates. The enzyme of interest is
manufactured by the cellular machinery. Alternatively, the
invention also allows for the screening of other secreted
biomolecules, including cytokines, chemokines, antibodies, and
metabolites, in solution for a desirable phenotype.
[0039] Evaluation of libraries of genetic variants of biomolecules,
such as enzymes, to identify specific members in the library with
desired properties (catalytic rate, specificity of reaction,
substrate binding affinities) requires both characterizing the
phenotype of the biomolecule produced and correlating the
biomolecule to the genotype of the member of the library encoding
it. In this way, desired variants can be selected and further
evaluated. Correlating the phenotype of the biomolecule and the
genotype of the producing cell is challenging. The invention
provides a method for isolating particular members from a library
of variant cells in individual microreactors, wherein the phenotype
of the biomolecule encoded by the cell is evaluated on the basis of
multiple parameters, including substrate specificity and kinetic
efficiency. The spatial segregation of the library members allows
each to be evaluated in parallel, and members exhibiting desired
characteristics are subsequently retrieved for further analysis
from the microreactor. A significant application of the technology
is the directed evolution of diverse enzymes for use in the in
vitro construction of biomolecules. One example is a method for the
identification of mutant GTases to transfer sugars from activated
donor molecules to the appropriate acceptor with absolute chemical
control. Such enzymes are capable of competing with chemical
synthesis for the rapid and large scale production of complex
carbohydrates.
[0040] When evolving enzymes from a library of enzyme variants, a
simple strategy to link a desirable phenotype to genotype is
necessary. The spatial separation of library members in individual
compartments allows the identification of variants with unique
properties without the requirement of substrate uptake or surface
attachment.
[0041] To that end, the invention described here uses
microfabricated chambers to separate a library of cells, e.g.,
yeast cells, which each secrete a mutant version of a protein of
interest (FIG. 1). The moldable slab, made of
poly(dimethylsiloxane), is fabricated by soft lithography and
replica molding and is of a biocompatible material, which is not
toxic and gas permeable. The rigidity of some materials, such as
polystyrene, would not allow for conformal contact, and thus
sealing, of the microwells against a substrate for testing the
specificity of the antibodies produced in a parallel. PDMS,
however, is a suitable material for this technique because it is
not toxic, it is gas permeable, and it is easily compressed to form
a tight, but reversible, seal with a rigid substrate. Such a seal
retards or to prevents any fluid and/or cells in the moldable slab
from leaking or escaping.
[0042] Cells confined in microwells and sealed against a glass
slide (such that the total media available was limited to the
volume of the microwell) are distributed at roughly one cell per
well in a device containing wells 50 .mu.m in diameter. FIG. 8C
depicts a protein microarray from single Pichia pastoris cells and
FIG. 8D shows the cells that secreted the protein microarray in 8C.
Pichia pastoris cells expressing a human Fc were grown in YPD media
overnight. Cells were then loaded into a Poly Dimethyl Siloxane
(PDMS) microdevice containing 50 .mu.m wells at roughly one cell
per well. The microdevice was contacted with a glass slide
pretreated with a goat anti-human Fc antibody to capture the
secreted Fc. The secreted proteins were captured over 90 minutes
and the resulting array was read using a Cy5-conjugated goat
anti-human Ig(H+L) antibody. The Pichia pastoris cells were imaged
in the microwells using a fluorescent dye for the yeast cell
surface. FIG. 9 shows how the secereted protein levels for Pichia
pastoris compare to other cell types, such as hybridomas, and
cytokine-secreting peripheral blood mononuclear cells (PBMC). This
standard curve was created using purified human Fc, and the
intensity values observed were used to assign defined
concentrations to the secretions captured from individual cells.
The amount of secreted proteins observed for Pichia pastoris cells
is well above the limit of detection for the assay and should
provide adequate concentration levels in microwells for the
turnover of supplied enzyme substrates. These experiments
demonstrate the ability to detect secreted products from individual
yeast cells. See also, Love et al., 2006 Nat. Biotechnol,
24(6):703-707; WO 2007/035633.
[0043] In a particular example, a library of segregated yeast cells
is interrogated with enzyme substrates yielding a fluorescence
signal upon successful enzyme turnover. Since the intensity of
signal correlates directly with product formation, library members
are directly compared for enzyme kinetics in addition to substrate
specificity via real-time fluorescence monitoring. Clones from
fluorescent wells are retrieved using micromanipulation and used in
further rounds of evolution and selection. Cell retrieval using a
micromanipulator is shown in FIG. 10. Yeast survivability following
retrieval with a micromanipulator was 40-60%.
Mutagenesis Techniques for Improving Enzymes
[0044] Mutations that encode amino acid changes are useful for
generating novel enzyme activities. The genes are obtained using
any method known to one of skill in the art, e.g., by isolating
clones from a genomic library of a given organism, by polymerase
chain reaction (PCR) amplification from a source of genomic
deoxyribonucleic acid (DNA) or messenger ribonucleic acid (mRNA),
or from a library of expression clones from a heterogeneous mixture
of DNA from uncultivated environmental microbes (U.S. Pat. No.
5,958,672). There are numerous methods that are well known to those
skilled in the art for mutating the genes encoding enzymes and
other non-catalytic proteins and peptides. These methods include
both rational (e.g., creating point mutants or groups of point
mutants by site-directed mutagenesis) and stochastic (e.g., random
mutagenesis, combinatorial mutagenesis and recombination)
techniques. A rational design, termed protein design automation,
uses an algorithm to objectively predict protein sequences likely
to achieve a desired fold.
[0045] One class of techniques is those relying on point mutations,
e.g., error-prone polymerase chain reaction and
oligonucleotide-directed mutagenesis (Cadwell and Joyce, 1992 PCR
Methods Applic., 2:28-33; Kegler-Ebo D M, et al., 1994 Nuc Acids
Res, 22(9):1593-1599). These methods lead to the production of an
enzyme library that contains members having any of the 20 different
amino acids at one specific position within a given protein.
[0046] Stochastic methods include, for example, chemical
mutagenesis (Singer and Kusmierek, 1982 Annu Rev Biochem,
51:655-93), recursive ensemble mutagenesis (Arkin and Youvan, 1992
Proc Natl Acad Sci USA, 89(16):7811-5; Delagrave et al., 1993
Protein Eng, 6(3):327-31), exponential ensemble mutagenesis
(Delagrave and Youvan, 1993 Biotechnology, 11(13):1548-52),
sequential random mutagenesis (Chen and Arnold, 1991 Biotechnology,
9(11):1073-7; Chen and Arnold, 1993 Proc Natl Acad Sci USA,
90(12):5618-22), DNA shuffling (Stemmer, 1994 Proc Natl Acad Sci
USA, 91(22):10747-51; Stemmer, 1994 Nature, 370(6488):389-91) and
the like. Recombination is a useful stochastic mutagenesis
technique wherein DNA is broken down and rejoined in new
combinations. DNA shuffling, the best known method of
recombination, allows useful mutations from multiple genes to be
combined (Stemmer W P C, et al., 1994 Nature, 370:389-391.)
Staggered extension process (StEP) is a simple and efficient method
for in vitro mutagenesis and recombination of polynucleotide
sequences (Zhao H, et al., 1998 Nature Biotechnol, 16:258-261.)
Other mutagenesis techniques include chemical mutagenesis and the
use of mutator strains (Lai Y, et al., 2004 Biotech Bioeng,
86:622-627; Coia G, et al., 1997 Gene, 201:203-209). These
techniques are used individually or in combination to produce
mutations.
[0047] DNA encoding the desired enzyme or protein is isolated from
the expression library and sequenced. By repeating the steps of
mutagenesis and screening, novel enzymes and other proteins are
artificially created. This iterative process is known as directed
evolution. The genes of interest do not necessarily have to be
expressed on plasmids. In one aspect, they are expressed following
integration into the host chromosome or as a result of mutating the
chromosomal copy of a gene. In another aspect, high complexity
expression libraries are created without mutagenesis. This can be
done by cloning and expressing DNA from a source that already
contains a large number of different sequences, such as highly
heterogeneous genomic DNA from a mixture of environmental
microbes.
Activity Screening of Expression Libraries
[0048] The methods described by the invention allow for the
biomolecule to be assayed for function. In one aspect, screening
for the desired biological activity is performed using aptamers,
i.e., oligonucleic acid or peptide molecules that bind a specific
target molecule. In another aspect, screening for the desired
biological activity is performed using a solution-phase FRET-based
assay in the microwells of the microdevice with fluorogenic
substrates. In another aspect, biological activity is assayed via
solid-support fluorescence (or FRET), wherein substrates/products
are captured on the contacted glass surface using antibodies.
Preferably, one or more of the substrates are fluorescent. In yet
another aspect, screening for the desired biological activity is
performed via solid-support affinity capture, wherein one or more
substrates are further derivitized with a fluorophore using a
chemical or enzymatic reaction (i.e., "click chemistry", sortase
tagging, BirA biotinylation, etc.). Alternatively, the function of
the biomolecule is assayed using a solid-support antibody-based
fluorescence readout, wherein both substrates have affinity tags
and the product is detected in a sandwich ELISA format. Preferably,
the secondary antibody is conjugated to a fluorophore.
[0049] In one aspect, screening for the desired biological activity
is done by contacting the host cells expressing the enzyme with a
chromogenic or fluorogenic compound that is appropriate for the
enzyme reaction and monitoring the formation of color in the cells
or their surroundings. In the solid-phase assays described in U.S.
Pat. No. 5,914,245, these compounds are referred to as optical
signal substrates because they produce a measurable change in
absorbance, reflectance, fluorescence or luminescence when they
come in contact with active enzyme or with a product of the
enzymatic reaction.
[0050] The invention provides for various chromogenic, fluorogenic,
lumigenic and fluorescence resonance energy transfer (FRET)
substrates to measure biological activity. Typically, fluorophores
absorb electromagnetic energy at one wavelength and emit
electromagnetic energy at a second wavelength. Representative
fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS;
4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;
5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;
5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM
(5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy
Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA
(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G;
6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine;
ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine);
Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;
Acriflavin Feulgen SITSA; Aequorin (Photoprotein);
AFPs--AutoFluorescent Protein--(Quantum Biotechnologies) see sgGFP,
sgBFP; Alexa Fluor 350.TM.; Alexa Fluor 430.TM.; Alexa Fluor
488.TM.; Alexa Fluor 532.TM.; Alexa Fluor 546.TM.; Alexa Fluor
568.TM.; Alexa Fluor 594.TM.; Alexa Fluor 633.TM.; Alexa Fluor
647.TM.; Alexa Fluor 660.TM.; Alexa Fluor 680.TM.; Alizarin
Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA
(Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;
Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC
(Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red
4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL;
Atabrine; ATTO-TAG.TM. CBQCA; ATTO-TAG.TM. FQ; Auramine;
Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole);
BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta
Lactamase; BFP blue shifted green fluorescent protein (GFP) (Y66H);
Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzamide;
Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV;
BOBO.TM.-1; BOBO.TM.-3; Bodipy 492/515; Bodipy 493/503; Bodipy
500/510; Bodipy 505/515; Bodipy 530/10; Bodipy 542/563; Bodipy
18/568; Bodipy 564/517; Bodipy 576/589; Bodipy 581/591; Bodipy
630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FI; Bodipy FL
ATP; Bodipy FI-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X
conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X
SE; BO-PRO.TM.-1; BO-PRO.TM.-3; Brilliant Sulphoflavin FF; BTC;
BTC-5N; Calcein; Calcein Blue; Calcium Crimson.TM.; Calcium Green;
Calcium Green-1 Ca.sup.2+Dye; Calcium Green-2 Ca.sup.2+; Calcium
Green-5N Ca.sup.2+; Calcium Green-C18 Ca.sup.2+; Calcium Orange;
Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue.TM.;
Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP--Cyan
Fluorescent Protein; CFP/YFP FRET; Chlorophyll; Chromomycin A;
Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp;
Coelenterazine f; Coelenterazine fcp; Coelenterazine h;
Coelenterazine hcp; Coelenterazine ip; Coelenterazine n;
Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM
Methylcoumarin; CTC; CTC Formazan; Cy2.TM.; Cy3.1 8; Cy3.5.TM.;
Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.; Cyan GFP; cyclic AMP
Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl
Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride;
4',6-diamidino-2-phenylindole (DAPI); Dapoxyl; Dapoxyl 2; Dapoxyl
3'DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR
(Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA
(4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH);
DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydrorhodamine 123
(DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR
(DiIC18(7)); DM-NERF (high pH); 2,4-Dinitrophenol (DNP); Dopamine;
DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97;
Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium
homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)
chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF
(Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3;
Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;
Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM
1-43.TM.; FM 4-46; Fura Red.TM. (high pH); Fura Red.TM./Fluo-3;
Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant
Yellow IOGF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer
(CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV
excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;
Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258;
Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin;
Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high
calcium; Indo-1, low calcium; Indodicarbocyanine (DiD);
Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;
LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;
Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine
Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer
Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker
Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;
LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red
(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1;
Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue;
Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF;
Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker
Orange; Mitotracker Red; Mitramycin; Monobromobimane;
Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green
Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole;
Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant
lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green.TM.;
Oregon Green.TM. 488; Oregon Green.TM. 500; Oregon Green.TM. 514;
Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7;
PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red);
Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine
3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26
(Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3;
PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid
(PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin
7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin;
RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine
5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B
extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine
Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT;
Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A;
S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant
Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron
Orange; Sevron Yellow L; sgBFP.TM.; sgBFP.TM. (super glow BFP);
sgGFP.TM.; sgGFP.TM. (super glow GFP); SITS; SITS (Primuline); SITS
(Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2;
SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen;
SpectrumOrange; Spectrum Red; SPQ
(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine
B can C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14;
SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO
23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44;
SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO
80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX
Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC);
Texas Red.TM.; Texas Red-X.TM. conjugate; Thiadicarbocyanine
(DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin
S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS
(Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1;
TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelso
ThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC;
wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H;
Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, Sybr
Green, Thiazole orange (interchelating dyes), semiconductor
nanoparticles such as quantum dots, or caged fluorophore (which can
be activated with light or other electromagnetic energy source) or
a combination thereof.
[0051] A wide variety of suitable donor (D) and acceptor (A)
fluorophores suitable for use in FRET are commercially available.
The choice of probe pair is influenced by system constraints as
well as by the length and sequence of the peptide used in the
desired application. The length and sequence of the peptide will
influence the labeling sites for attachment of the probes. The
distance between the attachment sites influences the choice of the
donor/acceptor pair due to the distance-dependence of FRET. Many
donor/acceptor pairs are commercially available. These include, but
are not limited to: 5-TAMRA/QSY-7; Dansyl/Eosin; Tryptophan/Dansyl;
Fluorescein/Texas Red (rhodamine); Naphthalene/Dansyl; Dansyl/ODR;
BODIPY/BODIPY; Terbium/Thodamine; Dansyl/FITC; Pyrere/Coumarin;
IAEDANS/IAFBPE/Cy5; and Europium/Cy5. A biotin or other small
affinity tag is used in detection of the protein via anti-biotin
antibodies or avidin/streptavidin tagged detectors like horseradish
peroxidase or a fluorescent dye.
[0052] In one aspect, indicator compounds are used to detect one or
more products of an enzymatic reaction by interacting either
directly or indirectly with the products. Optionally, these
indicator compounds are included as part of the optical signal
substrate solution. For example, U.S. Pat. No. 5,914,245 describes
a lipase assay that detects fatty acid interactions with the
fluorescent dye Rhodamine B. Other assays that can utilize
indicator compounds include those wherein protons are generated or
wherein transmembrane proton, electron or ion transfer occurs
during an enzymatic reaction. These activities can be detected by
including various dyes in the substrate solution. Fluorescein
isothiocyanate (FITC) is a derivative of fluorescein used in
wide-ranging applications including flow cytometry. Exemplary
fluorescent indicator dyes used to monitor pH changes include
fluorescein and seminaphthorhodafluors and their derivatives for
the pH range 6-9 and LysoSensor, Oregon Green and Rhodol and their
derivatives for the pH range 3-7. These fluorescent pH indicators
are available from Molecular Probes (Eugene, Oreg.). Chromophore
dyes whose wavelength of maximum absorption changes as a function
of pH include Thymol Blue (approximate useful pH range 1.2-2.8 and
8.0-9.6), Methyl Orange (pH 3.2-4.4), Bromocresol Green (pH
3.8-5.4), Methyl Red (pH 4.2-6.2), Bromothymol Blue (pH 6.0-7.6)
and Phenol Red (pH 6.8-8.2). Phenolphthalein (pH 8.2-10.0) turns
from colorless to pink as the pH becomes more alkaline. These
colorimetric pH indicators are available from Sigma-Aldrich (St.
Louis, Mo.). There are numerous examples in enzymology of using pH
indicators for detecting enzymatic activity (Lowry et al., 1951 J
Biol Chem, 193:265-275; Khalifah, 1971 J Biol Chem,
246(8):2561-73). Indicators such as Bromothymol Blue and Phenol Red
have been used to assay the activity of various hydrolases in
solution (Moris-Varas et al., 1999 Bioorg Med Chem,
(10):2183-8).
Mucin-Type O-Linked Glycosylation
[0053] The most abundant form of O-linked glycosylation in higher
eukaryotes is known as "mucin-type" (Hang H and Bertozzi C, 2005
Bioorg Med Chem, 13(17):5021-5034). The first step in mucin
biosynthesis is .alpha.-N-acetylgalactosamine (GalNAc) addition to
hydroxyl groups of serine or threonine side chains to form the
Tn-antigen; this transfer is accomplished by the polypeptide
N-acetyl-.alpha.-galactosaminyltransferases (ppGalNAcTases) (Ten
Hagen et al., 2003 Glycobiol, 13(1):1R-16R). The Tn-antigen is
elaborated further by downstream GTases to produce a variety of
mucin-type structures (FIG. 2). To date, over 150 glycoproteins
containing mucin-type glycosylation have been identified, many of
which are involved in disease progression (Hang 2005). One such
example is MUC1, a glycoprotein that has been identified as a tumor
antigen due to its increased expression in cancer epithelial cells,
which contributes to both cancer cell adhesion and tumor
invasiveness (Yu et al., 2007 J Biol Chem, 282(1):773-781; Kohlgraf
et al., 2003 Cancer Res, 63(16):5011-5020). Cancer-associated
mucins are highly immunogenic and may be used as targets for
immunotherapy (Hanisch and Ninkovic, 2006 Curr Prot Pep Sci, 7:307;
Tarp and Clausen, In Press Biochem Biophys Acta).
Synthesis of Homogeneous Mucin-Type O-Linked Glycopeptides and
Glycoproteins
[0054] The development of carbohydrate vaccines requires access to
large quantities of homogeneous glycopeptides and glycoproteins
(Grogan et al., 2002 Annu Rev Biochem, 71:593-634). The isolation
of native or recombinant glycoproteins, however, only yields
limited amounts of heterogeneous glycoforms, each of which can
display different biological properties (Freire et al., 2006
Glycobiol, 16(11):1150). Chemical synthesis of glycoconjugates
provides homogeneous substrates via solid-phase peptide synthesis
(SPPS) using an appropriately protected glycosyl amino acid
building block (Marcaurelle and Bertozzi, 2002 Glycobiol,
12(6):69R-77R). Native chemical ligation and expressed protein
ligation have also been used to install sugars site-specifically in
larger peptides and even proteins (Muir T W, 2003 Annu Rev Biochem,
72:249-289). Prior to the invention described herein, accomplishing
these synthetic methods still required a specially trained chemist.
The invention provides for the generation of enzymes capable of
efficient synthesis of glycoconjugates on a preparative scale,
which greatly aids in their study for therapeutic purposes.
GTase Evolution for the Synthesis of Carbohydrate-Containing
Natural Products.
[0055] The identification of glycoproteins and glycolipids that are
overexpressed on the surfaces of cancer cells has led to their
investigation as targets for immunotherapy (Slovin et al., 2005
Immunol Cell Biol, 83(4):418). As tumor-associated carbohydrate
antigens are typically expressed in low levels and in various
glycoforms, the isolation of sufficient amounts of discrete
glycoconjugates for developing carbohydrate-based anticancer
vaccines is difficult. Prior to the invention described herein,
general methods for the chemical synthesis of carbohydrates have
improved with the advent of automated assembly (Plante et al., 2003
In: Advances in Carbohydrate Chemistry and Biochemistry, Vol. 58,
pp 35-54), but still require a specialist to accomplish the
extensive protecting group manipulations requisite for
stereochemical control and donor/acceptor compatibility. Additional
shortcomings of the chemical synthesis of glycoconjugates include
the difficulty in generating large scale amounts to meet clinical
requirements and the difficulty in purifying the synthesized
materials.
[0056] Nature efficiently makes carbohydrate-containing compounds
using glycosyltransferases (GTases) to transfer sugars from
activated donor molecules (e.g., UDP sugars) to the appropriate
acceptor (e.g., proteins/peptides, lipids, other sugars and natural
product aglycones--polyketides and macrolides) with absolute
chemical control/stereochemistry. The following GTases glycosylate
diverse acceptors using three different donors, yet have a very
similar fold: GtfB--glucose transfer to vancomycin aglycone, BTG
.beta.-glucosyl transferase, and MurG-GlcNAc transfer in cell wall
biosynthesis.
[0057] While most GTases are highly substrate selective, relatively
few structural motifs are used to glycosylate a wide range of
glycosyl acceptors (Hu Y and Walker S, 2002 Chem Biol,
9:1287-1296). The invention provides for directed evolution of
existing GTases to identify more potent catalysts with altered
substrate selectivity. More specifically, the invention provides
for the identification of mutant GTases capable of competing with
chemical synthesis for the rapid and large scale production of
glycoconjugates for therapeutic purposes, including
carbohydrate-based cancer vaccines.
[0058] Engineered GTases have enormous potential for the synthesis
of biologically relevant glycoconjugates, either by improving the
catalytic efficiency of native glycosylation or by incorporating
non-natural sugar residues (Hancock et al., 2006 Curr Opin Chem
Biol, 10(5):509-519). However, prior to the invention described
here, few attempts had been made to engineer GTases by directed
evolution, largely due to the lack of methods for screening and
selecting mutants on the basis of GTase activity. Recent examples
include the engineering of a sialylotransferase (Lairson et al.,
2006 Nat. Chem. Biol., 2(12):724-728) and a glucotransferase
(Williams et al., 2007 Nat. Chem. Biol., 3(10):657-662), but the
generality of the screening methods used in these cases is unclear.
The first method requires fluorescent substrates to be ingested by
competent clones to sort them by flow cytometry, and the second
method uses the fluorescent molecules themselves as the aglycone
acceptors. The invention described here provides a general strategy
that allows for the ex vivo screening of diverse enzymes using
native or minimally perturbed substrates.
[0059] M13 phage display is a convenient strategy to link phenotype
and genotype in the engineering and selection of enzymes that do
not provide cell-based phenotypes (Hoess R, 2001 Chem Rev,
101:3205-3218). In phage-display enzyme evolution, enzymes and
substrates are proximally bound on the surface of phage to enable
deconvolution of the library by affinity capture of the products.
Recently, a chemically straightforward method was developed for the
attachment of substrates to the surface of phage using
selenocysteine residues (Love et al., 2006 Chembiochem,
7(5):753-756). In that study, the bacterial GTase MurG was
expressed on phage in active form; however, a successful evolution
of MurG was unsuccessful due to the inability of phage-bound enzyme
to utilize phage-bound substrate. The new technique provided by the
invention extends the method to eukaryotic enzymes and provides
improved methods of screening a library of mutant GTases.
[0060] One advantage of the methods described by the invention is
that neither the enzymes to be assayed, nor the substrates for
those enzymes need to be attached to any type of solid support,
e.g., a solid surface, another cell, etc. Moreover, the methods of
the invention are performed in solution with secreted biomolecules.
The invention provides for screening biomolecules secreted from
individual cells, instead of from microcolonies, which are clumps
of cells. Additionally, cells secreting active clones are retrieved
from the device by micromanipulation with a glass capillary, and
then either mutagenized randomly for further selection or sequenced
to identify the encoded enzyme.
[0061] Another distinguishing characteristic of the methods
described by the invention is that multiple characteristics of each
library member are assessed during the screening process. Unlike
surface-display methods on phage, bacterial cells or yeast, the
rates of enzymatic turnover can be monitored in real-time in the
microreactors on the basis of changes in the measured fluorescent
intensities. Competitive assays using two substrates modified with
different fluorophores allows direct monitoring of substrate
specificity or selectivity during the screening. These measurements
provide a greater degree of diversity in the clones identified and
selected for further rounds of evolution than existing
techniques.
Applications
[0062] Biocatalysis is an important tool for the synthesis of bulk
chemicals, pharmaceuticals and food ingredients. The number and
diversity of such applications are limited, however, likely due to
limitations in enzyme stability, catalytic properties, i.e.,
turnover rate, and substrate scope. Access to a tool kit of
biocatalysts will help industry overcome the current limitations
and enable the realization of many new applications, from
single-step enzymatic conversion to multi-step microbial synthesis
via metabolic pathway engineering.
[0063] The biosynthesis of carbohydrate-containing natural products
is of particular interest in industry, as their synthesis by
traditional means requires lengthy protecting group manipulations
and studies in glycosyl donor/acceptor compatibility. Therapeutic
vaccines derived from glycoprotein or glycolipid constructs that
are overexpressed on the surfaces of malignant cells are a
promising approach for cancer immunotherapy.
Synthesis of Novel Macrolide Antibiotics
[0064] The increasing incidence of antibiotic resistant bacterial
infections indicates the need for improved constructs to treat
enterococcal-infected patients. Many macrolide and polyketide
antibiotics contain carbohydrates that participate in recognition
of a cellular target and are thereby essential for activity (Walsh
C, 2003 Antibiotics: Actions, origins, resistance. 1.sup.st ed.;
American Society for Microbiology Press: Washington, D.C.).
Modification of existing glycopeptide antibiotics, such as
vancomycin and teicoplanin, on and around the sugar substitutents
has led to the clinical trials of new treatments, including
oritavancin (Dong et al., 2002 J Am Chem Soc, 124:9064-9065).
Adaptation of GTases as catalysts for the attachment of diverse
carbohydrates to natural product aglycones, proteins and lipids
will provide new materials for investigation as therapeutic agents.
The methods provided by the invention identify enzymes capable of
efficiently glycosylating a range of substrates and segue into the
generation of catalysts able to compete with chemical synthesis for
the rapid and large scale production of glycoconjugates.
Example 1
The Development of a New Technique for Screening a Library of
Mutant Enzymes for Improved Catalytic Activity or Altered Substrate
Specificity
[0065] The following experiment consists of (1) illustration of a
technique for the spatial separation of a library of yeast cells
secreting an enzyme of interest, and (2) enrichment of cells
expressing an active protease from an inactive variant to determine
the sensitivity of the technique. Briefly, a library of yeast cells
capable of secreting a protein of interest is loaded into
microwells 50 microns in diameter so that each well contains, on
average, one library member. Each compartment in the device is
interrogated in parallel with enzyme substrates; successful enzyme
turnover yields a fluorescence signal. Feasibility of the technique
is demonstrated with a protease.
[0066] Microfabricated arrays of wells have been used for diverse
biological applications. Microwells have proven useful to study
enzymology at the single molecule level, and wells that are 50-100
.mu.m diameter have been used to separate cells to screen secreted
products captured on a surface (Rondelez et al., 2005 Nat
Biotechnol, 23(3):361-365; Love et al., 2006 Nat Biotechnol,
24(6):703-707). Microdevices of the latter sort contain
.about.100,000 wells on a footprint the size of a typical
microscope slide (1''.times.3'') making screening of a reasonably
sized library (10.sup.6 members) possible using 10 such devices in
one day on an optical microscope.
Selection of Expression Host
[0067] The invention provides for screening biomolecules secreted
by cells. In one aspect, the cells are prokaryotic cells.
Alternatively, the cells are eukaryotic cells. Preferably, the
eukaryotic cells are yeast cells. An exemplary yeast cell includes
Pichia pastoris. Yeast cells that secrete plasmid encoded proteins
are used for the expression of enzymes for evolution by means of
the methods of the invention. Eukaryotic expression hosts, such as
yeast, offer an advantage over bacterial expression for the
evolution of diverse enzymes, including the ppGalNAcTases, because
they contain the machinery necessary for proper protein folding,
secretion and post-translational modification. Yeast are also an
ideal size (-5-10 .mu.m in diameter) for spatial separation using
microdevices in a ratio of one cell per well, where each well is 50
.mu.m in diameter. Additionally, yeast divide rapidly making the
genotyping of a library member derived from a single cell possible
within hours.
[0068] Yeast cells capability to secrete encoded enzymes vary with
respect to cell cycle; yeast are most efficient at protein
secretion during the budding process. In one aspect, large
variations in secretion, or the inability of the yeast to secret a
particular protein of interest is circumvented using yeast surface
display. Yeast surface display has been useful in the evolution of
diverse antibodies and several active enzymes have been previously
displayed on the surface of yeast (Gai and Wittrup, 2007 Current
Opinion in Structural Biology, 17(4):467-473).
Validation of the Technique with a Model Enzyme
[0069] The feasibility of the devised enzyme selection strategy is
tested first with the 3C-type cysteine protease from tobacco etch
virus (TEV) (Malcolm B, 1995 Protein Sci, 4(8):1439-1445). Mutation
of the catalytic cysteine in TEV at residue 151 to alanine results
in a catalytically inactive variant (Phan et al., 2002 J Biol Chem,
277(52):50564-50572). Vectors containing the genes for native and
mutant species of TEV protease are mixed in various ratios
(1:10,000, 1:1000, 1:100, 1:10) and used to create a model library
for enrichment of the catalytically active species. Yeast cells
transformed with the vector mixture are segregated into wells as
outlined above (FIG. 1). Sensitivity of the assay is determined
using the optimum recognition site (ENLYFQG; SEQ ID NO: 1) for TEV
protease as part of a fluorescence resonance energy transfer (FRET)
substrate (option 1; FIG. 3) (Malcolm 1995; Behlke et al., 2005,
Fluorescence and fluorescence applications. Integrated DNA
Technologies). Peptide cleavage between the glutamine and glycine
residues disrupts the intramolecular FRET quenching and result in a
fluorescence signal.
Evolution of Catalytic Activity and Substrate Specificity
[0070] Following the successful enrichment of clones expressing
active catalysts, model experiments for the directed evolution of
the TEV protease are conducted. To further demonstrate the ability
to screen on the basis of catalytic activity, the inactive C151A
mutant is randomly mutagenized using error-prone PCR (polymerase
chain reaction) to recover catalytically competent variants. While
activity will likely be restored as a result of the direct
inversion of the mutation at residue 151, it is possible to
identify competent variants with alternate mutations. As the
ability to screen for enzyme kinetics is anticipated, it is
possible to identify clones with increased catalytic activity as
compared to wild-type TEV protease. Finally, a library of variants
constructed from mutagenesis of the wild-type TEV protease for
cleavage of a non-native substrate is examined (option 2 (2, X=Ala)
FIG. 3). After each round of selection, cells secreting active
clones are retrieved from the device by micromanipulation with a
glass capillary. Retrieved clones will either be randomly
mutagenized for further selection or sequenced to identify the
encoded enzyme.
Example 2
The Evolution of a Mutant GTase with Improved Catalytic
Activity
[0071] The following experiment consists of evolution of
ppGalNAcTase mutants with increased catalytic efficiency and
altered substrate specificity. Microdevices are used to screen for
mutants of ppGalNAcTase-T1 having improved catalytic efficiency.
ppGaINAcTase-T1 is responsible for the transfer of alpha-GalNAc to
Ser/Thr residues to form the Tn-antigen--a tumor-associated
carbohydrate epitope. Mutants identified in this screen are used
for the in vitro synthesis of the Tn-antigen.
[0072] A recent crystal structure of murine ppGaINAcTase-T1 shows
that this protein folds to form distinct catalytic and lectin
domains (Fritz et al., 2004 Proc Natl Acad Sci,
101(43):15307-15312). Error-prone PCR is used to create random
libraries of ppGalNAcTase-T1 mutagenized within the catalytic
domain. A library of transformants is spatially segregated as
previously described and screened using fluorescent substrates
(FIG. 4).
Design and Synthesis of Fluorescent ppGalNAcTase Substrates
[0073] A fluorescein-modified UDP-sugar donor along with a
TAMRA-modified peptide acceptor allows for product detection at 580
nm due to FRET between the two fluorophores following glycosylation
(Behlke 2005). Based on structural information about the UDP-sugar
binding pocket of ppGaINAcTase-T1 and other retaining GTases, a
UDP-GalNAc substrate (3) bearing fluorescein at C-2 is synthesized
as previously reported for UDP-GlcNAc (Fritz 2004; Patenaude 2002;
Helm et al., 2003 J Am Chem Soc, 125:11168-11169). Acceptor peptide
4 containing an optimized substrate sequence (GAGAFFPTPGPAGAGK; SEQ
ID NO: 2) for glycosylation by ppGalNAcTase-T1 is synthesized with
a C-terminal TAMRA using commercially available reagents (Gerken et
al., 2006 J Biol Chem, 281(43):32403-32416).
Confirmation of Activity in Retrieved Clones
[0074] Following adequate rounds of library selection and
amplification (typically 4-6), cells secreting active clones are
retrieved from the device by micromanipulation with a glass
capillary, and then either mutagenized randomly for further
selection or sequenced to identify the encoded enzyme. Encoded
enzymes are tested with the native, unmodified UDP-GalNAc and
peptide substrates to identify those best able to synthesize the
Tn-antigen in vitro. Capable library members are used to synthesize
Tn-antigen in large quantities for further study of its
immunological properties and potential use in developing anticancer
vaccines.
[0075] Secreted or surface-displayed enzymes may not be capable of
utilizing synthetic substrates containing bulky fluorophores
incorporated to assay enzyme function. In one aspect, the position
of the fluorophores within each substrate, particularly the
modified UDP-GalNAc, are changed until an accepted version is
achieved. Alternatively, azido-functionalized UDP sugars are
routinely employed to study glycosylation in vivo; the azide group
is a useful chemical tag for further derivatization and substrate
detection (Campbell et al., 2007 Molecular Biosystems,
3(3):187-194). In another aspect, the ppGalNAcTase acceptor peptide
is modified with biotin to allow for capture and subsequent
detection of coupled products with a lectin or antibody in a
sandwich-style assay. In one aspect, the biotin tag is used in
affinity chromatography together with a column that has avidin
(also streptavidin or Neutravidin) bound to it, which is the
natural chelator for biotin. Alternatively, this tag is used in
detection of the protein via anti-biotin antibodies or
avidin/streptavidin tagged detectors like horseradish peroxidase or
a fluorescent dye.
Evolution of a Mutant ppGalNAcTase with Altered Substrate
Preference
[0076] Structural studies of a retaining glycosyltransferase
closely related to ppGalNAcTase-T1 have shown that specific
residues of the enzyme contact moieties in the UDP-sugar donor (C-3
and C-4) to enhance specificity for UDP-GalNAc over UDP-GlcNAc
(Patenaude et al., 2002 Nat Struct Biol, 9(9):685-690; Fritz et
al., 2006 J Biol Chem, 281(13):8613-8619). Screening the library of
mutagenized TI variants described above with a fluorescein-modified
UDP-GlcNAc donor yields clones capable of transferring this
non-native substrate and improves the understanding of the
active-site specificity of GTases.
Extension to the Synthesis of Other Mucin-Type Glycoconjugates
[0077] The synthesis described above is extended by mutagenizing
sialyl transferase ST6GalNAc-1 to make the sialyl Tn-antigen (FIG.
2), using the in vitro synthesized Tn-antigen as a substrate.
Development of enzymes for the in vitro synthesis of various
mucin-type core structures enables the biological study of this
class of glycoconjugates, which have been implicated in a variety
of diseases.
Example 3
Enzyme Turnover in Microwells--Trypsin Cleavage Assay
[0078] The following experiment demonstrates detection of enzyme
activity in a cell-free microwell system. A method for detecting
enzyme turnover in microwells via a trypsin cleavage assay is
diagramed in FIG. 11. Increasing concentrations (0.05 .mu.g/ml, 0.5
.mu.g/ml, and 5 .mu.g/ml) of trypsin were incubated with 10
.mu.g/ml FTC-casein for 1 hour in microwells. As shown in FIG. 12,
the intensity of the observed fluorescent signal was dependent on
the concentration of trypsin in the microwells. In a separate
experiment, 0.5 .mu.g/ml of trypsin was incubated with 10 .mu.g/ml
FTC-casein in microwells, and photomicrographs were taken at 1 and
18 hours. As shown in FIG. 13, the intensity of the observed
fluorescent signal was dependent on the time of incubation.
Microwells have been used previously to study isolated enzymes in
microwells. See, JP2004309405A1; and Rondelez et al., 2005 Nat
Biotechnol, 23(3):361-365.
Example 4
Secreted Enzyme Turnover in Microwells--HRV-3C Protease Assay
[0079] The following experiment demonstrates that an enzyme, i.e.,
a protease secreted by individual Pichia pastoris (yeast) cells
inside the micro-device of the invention, cleaved a peptide
substrate with a FRET reporter pair, thereby identifying cells
containing active enzyme with a bright fluorescent signal.
Specifically, Pichia pastoris were genetically engineered to
secrete human rhinovirus 3C protease (HRV-3CP), which cleaved a
peptide substrate sequence (EDANS-A-L-E-V-L-F-Q/G-P-K-DABCYL; SEQ
ID NO: 3). A method for detecting enzyme turnover in microwells via
an HRV-3CP assay is diagramed in FIG. 14. Pichia pastoris capable
of secreting the HRV-3CP enzyme were loaded into the microdevice.
The cells were incubated in the microdevice for 18 hours in the
presence of the FRET peptide substrate
(EDANS-A-L-E-V-L-F-Q/G-P-K-DABCYL; SEQ ID NO: 3), supplied at 100
.mu.g/mL in YPD supplemented with 50 mM Tris, pH 7.0, 150 mM NaCl,
and 1 mM EDTA. The secreted enzyme successfully cleaved the
substrate, resulting in a fluorescent signal. The arrows in the
left panel of FIG. 15 point to cells in wells which correspond to
the bright fluorescent wells observed in the right panel of FIG.
15.
Sequence CWU 1
1
317PRTArtificial SequenceChemically Synthesized 1Glu Asn Leu Tyr
Phe Gln Gly1 5216PRTArtificial SequenceChemically Synthesized 2Gly
Ala Gly Ala Phe Phe Pro Thr Pro Gly Pro Ala Gly Ala Gly Lys1 5 10
1539PRTArtificial SequenceChemically Synthesized 3Ala Leu Glu Val
Leu Phe Xaa Pro Lys1 5
* * * * *